Self-assembling tissue modules

ABSTRACT

The disclosure relates to a new approach to constructing cellular aggregates in vitro and their use in methods for producing 3D-tissue constructs in a modular way. In particular, the disclosure is directed to a method for in vitro producing a tissue construct comprising: a) combining living cells to form supracellular aggregates using spatial confinement; b) combining two or more of the supracellular aggregates in a mold or on a biomaterial; c) applying conditions that induce self-assembly within the combined supracellular aggregates to obtain the tissue construct; and d) applying conditions that induce tissue morphogenesis in the tissue construct.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/999,841, filed Mar. 14, 2011, pending, which is a national phaseentry under 35 U.S.C. §371 of International Patent ApplicationPCT/NL2009/050368 filed Jun. 22, 2009, designating the United States ofAmerica and published in English as International Patent Publication WO2009/154466 on Dec. 23, 2009, which claims the benefit under Article 8of the Patent Cooperation Treaty to European Patent Application SerialNo. 08158652.1 filed Jun. 20, 2008, the disclosure of each of which ishereby incorporated herein in its entirety by this reference.

TECHNICAL FIELD

The disclosure relates to a new approach to constructing cellularaggregates of defined sizes and shapes in vitro and their use in methodsfor producing 3D-tissue constructs in a modular way.

BACKGROUND

Most tissues consist of multiple cell types organized with specificmicroscale heterogeneity. Typically, one cubic centimeter can hold up to300 million cells. These cells form different structures within thetissue, including blood capillaries and a neural network, which arecrucial for nutrition, innervation and homeostasis of the tissue. Cellsorganize and interact in a multitude of architectures and synthesize avariety of biologically active molecules for mechanical support andcellular instruction. Therefore, living tissues are highly complex.

Tissue engineering is a term used for attempts to produce living tissuein vitro from individual or groups of cells. It aims at repairing orreplacing portions of or whole tissues and provides solutions toshortage of organ donation or to the use of experimental animals fortesting new therapies.

Due to the high complexity of living tissue, efforts to produce or mimicliving tissues in vitro have been in vain to date and new methods andtechnologies to assemble cells into tissue structures are needed. Thisis currently a main limitation in disciplines like regenerativemedicine, pharmaceutics, oncology and developmental biology in which 2Dculture and crude small 3D cell aggregates (see, e.g., WO-A-00/75286)are still standards. As a result, biological in vitro experiments do noteven come close to complex biological reality, research progress isseverely inhibited, and experimental animals have to be used as anunsatisfactory experimental alternative instead.

With the recent developments in both adult and embryonic stem cellbiology, it is becoming truly feasible to induce cells in culture intomore and more of the individual cell types that are found in the humanbody and in spectacular high numbers.

Unfortunately, a satisfactory technology to go from a large pool ofcells including different cell types to a tissue mimic with a complexarchitecture has not yet been developed. Several possible strategies,such as organ printing (see, e.g., WO-A-2005/081971) and cell sheettechnology, are currently being explored. These strategies rely heavilyon the possibility of positioning (pools of) cells in a predefinedorganization. These strategies are encountering obstacles that preventthe translation of a complex architecture to an actual centimeter scaletissue (i.e., remodeling of the tissue construct over time due tophysical shrinkage or cell migration). Furthermore, the rationale behindorgan printing is still beyond reach of contemporary science as itsimply requires too many (10⁸) single steps. Even at a currentlyunattainable speed of depositing one thousand individual cells persecond at the correct three-dimensional location with micrometeraccuracy, it would take close to four days to build a single cubiccentimeter of tissue. These approaches and related technologies resultin a metastable multicellular construct: the construct is not stable butwill remodel according to complex biological principles. This means thatwith these strategies, a designed structure and complexity is nottranslated to the objective tissue. Promoting the self-assembly andself-organization of pools of cells is thus a more powerful approach. Inthis approach, cells are assembled into a construct prone to apredictable remodeling over time. Under appropriate boundary conditions,the construct leads to a final organized tissue. This is achieved byusing a bottom-up approach to sequentially assemble cells into,subsequently, supracellular aggregates and tissues and by promoting theself-organization of the tissue using boundary conditions.

Several attempts to assemble cells into tissues using a bottom-upapproach are already described, which are different from the presenteddisclosure. McGuigan and Sefton (PNAS 2006, 103 (31), 11461-11466) haveundertaken an attempt to overcome these practical difficulties bystarting from microscale modular components consisting ofsubmillimeter-sized collagen gel rods seeded with endothelial cells intoa micro-vascularized tissue. These modules were manually assembled intoa larger tube and perfused by medium or blood. However, their approachrequires the use of a gel, in this case a collagen gel, to obtain themodules and retain their structural integrity during the subsequentmanual assembly into larger structures. Although the use of a gel can beadvantageous in some cases to control, for instance, cell density, theentrapment of cells within a gel will decrease the plasticity of themodules and prevent fusion between modules. Eliminating the necessity touse gels for the formation of tissue modules allows for more plasticityand physiological remodeling of the tissue during the self-assemblyprocess. Sodunke et al. (Biomaterials 2007, 28 (27), 4006-4016) describea similar approach based on a biomatrix hydrogel. Gels have thedisadvantages in that the interface is not available and in that thecells have low movability.

An early attempt to generate gel-free cellular aggregates for use asbuilding blocks to construct bigger tissues has been described by Kelmet al. (Tissue Eng. 2006, 12 (9), 2541-2553). This attempt is based onthe so-called “hanging drop”-method, wherein cells in an inverted dropof tissue culture medium precipitate and aggregate. However, this methodcannot generate sufficiently large numbers of cellular aggregates in ashort enough time. Conventional methods for producing multicellularmodels (such as the hanging drop method or micro-mass culture) sufferfrom a number of limitations including (i) a poor control of size andshape of the aggregates, and reproducibility, (ii) tedious andtime-consuming manipulations, and (iii) low production yield ofmicro-tissues. Napolitano et al. (Tissue Engineering 2007, 13 (8),2087-2095) describe a method to form cellular aggregates byself-assembly on micro-molded non-adhesive hydrogels. This document doesnot describe the formation of pre-condensed cellular aggregates in afirst step and subsequent self-assembly of the cellular aggregates asbuilding blocks in a second step. This method thus induces intense andnon-predictable remodeling (e.g., shrinking) of the tissue construct.

BRIEF SUMMARY

The disclosure aims at overcoming one or more of these problems byproducing supracellular aggregates of cells of any cell type usingspatial confinement. These aggregates are used as building blocks andcombined using boundary conditions promoting their self-assembly andself-organization to create complex multicellular architectures.

In a first aspect, the disclosure relates to a method for in vitroproducing a tissue construct comprising:

-   -   a) combining living cells to form supracellular aggregates using        spatial confinement;    -   b) combining two or more of the supracellular aggregates in a        mold or on a biomaterial;    -   c) applying conditions that induce self-assembly within the        combined supracellular aggregates to obtain the tissue        construct; and    -   d) applying conditions that induce tissue morphogenesis in the        tissue construct.

The disclosure provides various advantages over prior art methods,including the use of simple tools that can be handled in most biologylabs, the ability to generate a very large amount of aggregates in quickand simple procedures (e.g., 220,000 aggregates per conventional 12-wellplate), and the absence of a hydrogel as supporting material.

The spatial confinement can be achieved in various manners. A well-knownand often applied way is by using arrays of microwells. Other ways ofimposing spatial confinement include using air-liquid interfaces likethe Hanging drop method or microfluidic channels. Any biocompatible,processable material can be used for the spatial confinement applied forassembling the cells into supracellular aggregates.

The term “microwell” as used in this application is meant to refer to anarray of numerous cup-like structures formed in a substantially uniformlayer of material by photolithographic patterning, molding, embossing orother manufacturing processes. Each microwell thus includes a lower wall(which may be formed by a substrate on which the microwell material isdeposited) and one or more peripheral side walls (e.g., a singlecircular wall, or three or more contiguous substantially straight walls)that extend upward from the bottom wall and surround a predefined lowerwall area, with upper edges of the peripheral side walls defining anopen end of the microwell. Typically, microwells having an envelopingdiameter of 50-500 μm can be used. The depth of the microwells isnormally in the range of 100-1000 μm. For seeding, it is advantageousthat individual microwells are close to each other in order to preventcells staying on the spaces between the microwells. Thus, the maximumdistance between two individual neighboring microwells on the array canbe, for example, 300 μm or less, preferably 200 μm or less, morepreferably 100 μm or less, such as about 50 μm. The number of microwellsin the array can vary depending on the size of the microwells and thedistance between individual microwells. One array can suitably have50-20,000 wells, such as 50-5000, or 100-2000 wells.

The term “self-assembly” as used in this application is meant to referto the creation of tissue units (or small building units) by associationof individual cells or cellular aggregates. The individual cells orcellular aggregates adhere together in specific arrangements to giveone-dimensional, two-dimensional or three-dimensional superstructures.The aggregation may be spontaneous without human intervention, or may beas a result of changing local environmental conditions, e.g.,temperature, concentration of cells, physical boundaries (such asspecific shape or dimension of the microwells and/or mold), etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Top: illustrative scheme of the disclosure. Bottom: tools thatcan be used to bring the disclosure into practice.

FIG. 2. SEM picture of a microwell array (diameter 100 μm, depth 350 μm)for the spontaneous formation of micro-tissues. In insert, enlarged viewof wells molded in PDMS.

FIG. 3. Spheroids prepared from HUVECs cells in a PDMS microsystem(10:0.5) coated with 35,000 MW PEG at 50 mg/ml. Microwells are of 200 μmdiameter and 350 μm depth.

FIGS. 4A and 4B. Spheroids prepared from human bone marrow-derivedmesenchymal progenitor cells in a PDMS microsystem (10:0.5) coated with35,000 MW PEG at 50 mg/ml. Microwells are of 200 μm diameter and 350 μmdepth. FIG. 4A picture is taken just after seeding and picture of FIG.4B after two days of culture.

FIG. 5. Stainless steel mold (left) used as a master to replicate anagarose chamber (right) used to assemble aggregates.

FIG. 6. Aggregates of HUVEC and hMSC assembled into the agarose chamberjust after seeding after three days of culture. The width of the chamberis 1 mm; the depth of the chamber is 1 mm; and the length of the chamberis 1 cm.

FIGS. 7A and 7B. Self-assembly of aggregates of different cell types.Cross-section of cylinder described previously after three days (FIG.7A) and six days (FIG. 7B). On day 3, one can observe the segregation ofcell types with the HUVEC, in red, forming an exterior layer. After sixdays, the angiogenesis processes took place and capillaries are formedin the tissue construct.

FIGS. 8A-8F. Cellular aggregates of hMSC can be assembled into tissueconstructs of different shapes to build tissue units that can then beassembled into bigger constructs. FIGS. 8A and 8B: 15 minutes afterseeding the aggregates. FIGS. 8C and 8D: 5 hours after seeding theaggregates. FIGS. 8E and 8F: tissue constructs were released from thewells 24 hours after seeding the aggregates.

FIG. 9A. The size of the building blocks depends on the number of cellsseeded and on the size of the microwells.

FIG. 9B. Different compositions of culturing media induce differentlevels of compaction of the spheroids over time.

FIG. 9C. Different cell types show different plasticity and maintenanceof the shape over time.

FIG. 10. Remodeling of the tissue construct into a desired geometry byusing compensated shapes.

FIG. 11A. Local compaction of the tissue depends on the geometricalshape.

FIG. 11B. Local stress of the tissue depends on the geometrical shape.

FIG. 12. Tissues can further be assembled into centimeter-scale tissues.

DETAILED DESCRIPTION

In a first step, a method according to the disclosure comprisesproducing a supracellular aggregate of cells. These cells may be of thesame (“homocellular”) or different (“heterocellular”) type within oneaggregate. It is preferred, however, that one aggregate is formed ofcells of one cell type. Diversification of the tissue construct to beproduced may be achieved by combining aggregates of different celltypes.

Many cell types may be used to form the cell aggregates. In general, thechoice of cell type will vary depending on the type of three-dimensionalconstruct to be built. For example, for a blood vessel typethree-dimensional structure, the cell aggregates will advantageouslycomprise a cell type or types typically found in vascular tissue (e.g.,endothelial cells, smooth muscle cells, etc.). In contrast, thecomposition of the cell aggregates may vary if a different type ofconstruct is to be produced (e.g., intestine, liver, kidney, etc.). Oneskilled in the art will thus readily be able to choose an appropriatecell type for the aggregates, based on the objective type ofthree-dimensional construct. Non-limiting examples of suitable celltypes include contractile or muscle cells (e.g., striated muscle cellsand smooth muscle cells), neural cells, connective tissue (includingbone, cartilage, osteoblasts, osteoclasts, cells differentiating intobone-forming cells and chondrocytes, and lymph tissues), hepatocytes,cardiomyocytes, myocytes, Schwann cells, urothelial cells, parenchymalcells, epithelial cells (including endothelial cells that form liningsin cavities and vessels or channels, exocrine secretory epithelialcells, epithelial absorptive cells, keratinizing epithelial cells, andextracellular matrix secretion cells), and undifferentiated cells (suchas embryonic cells, progenitor cells, (mesenchymal) stem cells, bonemarrow cells, satellite cells, fibroblasts, and other precursor cells),among others. In addition, plant cells and algae may suitably be used.

The aggregates may vary in both size and shape. They may, for example,be in the form of a sphere, a cylinder (preferably with equal height anddiameter), a rod, a cube, or the like. Although other shaped aggregatesmay be used, it is generally preferable that the cell aggregates arespherical, cylindrical (with equal height and diameter), or cuboidal(i.e., cubes), as aggregates of these shapes may be easier tomanipulate. The shape of the cellular aggregates can play an importantrole in promoting self-assembly. Different shapes of aggregates cangenerate different arrangements by stacking. The shapes of the cellularaggregates can, for instance, promote close proximity between cellularaggregates (e.g., key-lock system), or create free space at theirinterfaces. Aggregates are substantially uniform in size andsubstantially uniform in shape when they are combined but differentshapes and sizes can be assembled to generate different heterogeneousstructures.

Although the exact number of cells per aggregate is not critical, itwill be recognized by those skilled in the art that the size of eachaggregate (and thus the number of cells per aggregate) is limited by thecapacity of nutrients to diffuse to the central cells, and that thisnumber may vary depending on cell type. Cell aggregates may comprise aminimal number of cells (e.g., two or three cells) per aggregate, or maycomprise many hundreds or thousands of cells per aggregate. Typically,cell aggregates comprise hundreds to hundreds of thousands of cells peraggregate.

The number of cells in one aggregate can be controlled by the appliedspatial confinement. For instance, the number of cells in one aggregatecan be dependent on the number of cells that are seeded in a microwelland the size of the well. There is, however, no 1:1 ratio, because celldeath and proliferation may occur during formation of the aggregate. Ina suitable embodiment, the number of cells that is provided permicrowell is 2-500,000, such as 100-100,000, or 100-50,000. Furthermore,the number of cells applied, e.g., per microwell, also depends on thedesired aggregate size.

For purposes of the present disclosure, the cellular aggregates aretypically from about 100 microns to about 600 microns in size, such asfrom about 200 to about 400 microns, although the size may be greater orless than this range, depending on cell type. In one embodiment, thecell aggregates are from about 250 microns to about 400 microns in size.In another embodiment, the cell aggregates are about 250 microns insize. For example, spherical cell aggregates are preferably from about20 microns to about 600 microns in diameter (such as from about 100microns to about 600 microns), cylindrical cell aggregates arepreferably from about 100 microns to about 600 microns in diameter andheight, and the sides of cuboidal cell aggregates are preferably fromabout 100 microns to about 600 microns in length. Aggregates of othershapes will typically be of similar size. The size of the aggregates canbe measured using standard light microscopy techniques.

The size of the cellular aggregates can be controlled by the spatialconfinement, such as by size of the microwells, as well as by the numberof cells that is used, such as the number of cells seeded to themicrowells. Importantly, the size of the aggregates depends more on thenumber of cells than on the enveloping diameter of the spatialconfinement. The size and/or the shape of the spatial confinement can beroughly adjusted to facilitate proper aggregate formation. If thespatial confinement is too large, the cells will not find each other andwill not aggregate. If the spatial confinement is too small, then notall cells will fit in the well. For example, for aggregates having asize between 0 and 90 μm, circular microwells with a diameter of 100microns are suitable; for aggregates having a size between 90 and 150μm, circular microwells with a diameter of 200 μm are suitable; foraggregates between 150 and 350 μm, circular microwells with a diameterof 400 microns are suitable.

As mentioned above, with a suitable embodiment of the disclosure,aggregates of cells are produced using arrays of microwells that can beproduced with technologies that include, but are not limited to:microchip technology, hot embossing, selective laser sintering, solidfree-form fabrication, and phase separation micro-molding. With thesetechnologies, arrays of microwells can be produced in sheets ofdifferent materials including, but not limited to: PDMS(polydimethylsiloxane), collagen, gelatin, hydrogels, and the like. Animportant advantage of the above-mentioned technologies is that they canproduce microwells with different size and shape.

The disclosure considers both the use of spatial confinement with singlemorphology (such as arrays containing single-microwell morphology) andspatial confinement with two or more morphologies (such as arrayscontaining two or more microwell morphologies). In an embodiment, cellaggregates are formed by applying a cell suspension on top of themicrowell array. Typically, the cell concentration in the cell seedsuspension is in the range of 500,000 cells per ml to 5,000,000 cellsper ml. Cells either settle in the microwells spontaneously due togravitational forces, or are forced in the microwells using, forinstance, centrifugal, capillary forces or microfluidic devices.

Upon spatial confinement, the cells will aggregate spontaneously byadhesion between the cells. The adhesion between the same cell types isnot necessarily better than between different cell types, although thismay be the case for some specific cell types. The adhesion between thedifferent cells differs from cell type to cell type. For instance, humanmesenchymal stem cells will form spheroids that condense a lot due tostrong adhesion between the cells, HUVEC will form spheroids that hardlycondense due to moderate adhesion between the cells, and Chinese hamsterovary cells will instead form plates of spheroids due to low adhesionbetween the cells. Assembly of the cells into supracellular aggregatesmay be assisted by various tools known in the art, such as microfluidictools, moving liquids, confining chambers with modular properties(adherent/non-adherent surfaces), using surfaces with topographies, orusing surfaces with coatings.

It is important to note in this aspect that in order for aggregates toform, the adhesion between cells and the surrounding material (such asthe material of the microwell) is preferably lower than the adhesionbetween the cells themselves. This can, for example, be achieved byusing microwells of materials that display low cellular adhesion, suchas PEG (polyethyleneglycol), PDMS or the like, or by coating themicrowell surface with molecules that prevent cellular adhesion, such asPEG and BSA (bovine serum albumin). Moreover, it is important to notethat the formation of aggregates depends on the cellular adhesion of thecell type used. When a certain cell type is unable to form cellularaggregates spontaneously, aggregation may be initiated using compoundssuch as fibronectin or collagen that can be added to the cellsuspension.

The shape of the aggregates can be manipulated by altering the spatialconfinement shape. The size of the aggregates can be manipulated byaltering the size of the spatial confinement, the cell concentration ofthe cell suspension used, and/or the composition of the culture mediumthat is used during cellular aggregation.

An advantage of using microwells when compared to classical methods toproduce cellular aggregates, like the hanging-drop method, is that in asingle “step,” one can make thousands of aggregates at the same time,instead of merely one aggregate. This enables the fabrication of thevast quantities of aggregates that are needed for this bottom-upapproach. For instance, in comparison with the spontaneous aggregationin a cell suspension, the microwells allow a precise control andreproducibility of the shape, size, and surface properties of theaggregates.

The disclosure is further illustrated in FIG. 1. The top schemedescribes the technical steps to assemble cells into tissues withgeometric steps in a bottom-up approach. Cells are assembled intospheroids that are used as building blocks to build tissues. Thesetissues are shaped, e.g., to promote self-remodeling and can beinfluenced to self-organize. For example, sharp tips of a triangulartissue promote compaction of the construct inducing furtherdevelopmental mechanisms. The bottom picture shows some of the toolsthat can be used to bring the disclosure into practice. Polymeric stampscan be used to replicate structures into agarose. Agarose chips can beinserted into a conventional microwell plate and used for cell andtissue culture.

The cell suspension can suitably be added to a container (for instance,12-well plate) in which an array of microwells has been placed on thebottom. The cells can then sink into the wells by gravitational orcentrifugational forces. In principle, the values of temperature and pHdo not have to vary from the values that are used during standardculture of the respective cell types. However, there are applicationsforeseeable where, for instance, a change in temperature can be used toinitiate cell aggregation. The basis for the cell suspension is normallya culture medium supplemented with standard nutrients (not differentfrom normal cell culture). Aggregation usually takes place in a standardincubator (humidified, 37° C., 5% CO₂). If different cell types areused, they can be mixed in one cell suspension, or they can be appliedseparately, depending on the initial situation one wants to create. Ifboth cells are mixed in one cell suspension, the different cell typeswill typically be regularly mixed in the resulting aggregate. If thedifferent cell types are applied in different cell suspensions one afterthe other, the resulting aggregate will typically consist of two (ormore) distinct regions containing the two (or more) different celltypes.

It is an advantage of the disclosure that aggregates may be formed thatdo not contain anything but living cells. In particular, the use of agel is not necessary. This way, aggregates of particular high celldensity may be formed. In some cases, this can lead to a better contactbetween the different cells for exchange of compounds, since some cellsrely on direct cell contact for cellular communication. In addition, theabsence of a gel allows for the cells to better produce their ownextracellular matrix in a physiological way. Furthermore, the additionof a gel from xenogenous origin may impose a complication for clinicalapplications. By only using autologous cells, the product is completelypatient-owned.

Step a) allows the condensation of cells into building blocks(supracellular aggregates). This condensation process that is occurringover time is essential, since shaped micro-tissues cannot be produced ona large (mm) scale by seeding the cells in large wells. Condensation ofthe small aggregates minimizes the condensation of the bigger shapes ina later stage. If the step of forming the supracellular aggregatesthrough condensation would be skipped, then the shape of a subsequentseeding surface (such as macro-wells) will not be translated to thedesired construct. After seeding, the tissue will condense toward aspheroid, regardless of the shape of the seeding surface. Apart fromthat, the inventors found that pre-condensation (i.e., formation ofsupracellular aggregates) allows seeding a larger number of cells asaggregates (e.g., spheroids), compared to seeding a cell suspension. Itis, therefore, necessary to first condense cells into dense buildingblocks (supracellular aggregates) that thereafter can be used andassembled into bigger constructs.

Thereafter, the cellular aggregates are combined to obtain larger tissueconstructs. This can be described as a two-stage process.

The first stage is the self-assembly of cellular aggregates into abigger tissue construct. In a suitable embodiment, the aggregates areremoved from a microwell array by flushing (culture) medium over thesurface of the microwell. Another possibility is to invert a chip withmicrowells onto the surface to be seeded. Aggregates are then releasedby gravitational or centrifugational forces and transferred to theseeding surface (e.g., biomaterial, scaffold, macro-well). Forself-assembly, the aggregates can, for instance, be transferred intowells having an enveloping diameter of at least 500 μm. Anybiocompatible, processable material can be used for the spatialconfinement applied for assembling the supracellular aggregates intotissue constructs.

Self-assembly of the cellular aggregates will be governed by imposed“boundary conditions” of the cellular aggregates, such as supracellularaggregate size, supracellular aggregate shape, supracellular aggregatesurface properties (for instance, hydrophilicity/hydrophobicity or acoating with bioactive molecules that results in specific interactionsbetween the cellular aggregates), supracellular aggregate electricalcharge, supracellular aggregate magnetic charge, and of “boundaryconditions” of the chamber used to assemble the cellular aggregates,such as adherent or non-adherent surfaces of the chamber, topographiesof the surface of the chamber, protein deposition and patterning at thesurface of the chamber and the use of microfluidic to promote thearrangement and assembly of the cellular aggregates.

Preferably, the “boundary conditions” are imposed on the aggregatesbefore they are released from the initial spatial confinement. Dependingon the type of boundary condition, this may or may not require an extraactive step. For instance, the boundary condition “supracellularaggregate size” is already imposed by the spatial confinement and theseeding density. The boundary condition “supracellular aggregate surfaceproperties” can be adjusted, for instance, by coating the aggregatesbefore releasing them from the spatial confinement. The boundarycondition “supracellular aggregate magnetic charge” can be imposedduring seeding (e.g., by including magnetic particles) or by coating theaggregates before releasing them from the spatial confinement.

After incorporating these boundary conditions to the cellular aggregatesor the chamber used for their self-assembly, self-assembly of thecellular aggregates can be guided, e.g., in a chamber or in a movingliquid by applying, for instance, mechanical constraints, shear stressusing a liquid, compression, shaking, electrical fields, magneticfields, or gradients of morphogens and/or growth factors. The shape,size and cell type(s) of the supracellular aggregates is important inthe early stage of the assembly to promote mesoscale organization andcreate the heterogeneous structure of interest. Self-assembly of theaggregates normally takes several hours. Typically, it takes at most oneday. The structure of interest can include the simple assembly ofspherical aggregates into the shape of a cylinder or the more complexassembly of spherical aggregates into blocks (cubes, triangles, etc.)that can then be assembled into bigger constructs. For example, usingthe plastic properties of cells, chambers with compensated shapes can bedesigned, which result in the desired tissue construct shapes. Thedesign and structures of those constructs should promote the creation oflocal environment leading to further remodeling and tissue development.

Some illustrative examples of conditions that can be used to promoteself-assembly of the cellular aggregates into a tissue construct includethe cell type, the medium used to culture the tissue, and the time ofincubation on, e.g., the microwell array before the transfer to thefinal chamber.

The second stage involves the remodeling and/or reorganization of thecells and/or tissue in the construct. In this stage, conditions areapplied that induce tissue morphogenesis in the tissue construct. Theterm “morphogenesis” in this application is meant to refer to acoordinated series of molecular and cellular events that shape thestructure of the tissue construct. Tissue morphogenesis and can begoverned by migration of cells, physical traction of cells,differentiation of cells, local production of soluble or insoluble(extra-cellular matrix) biological factors, or combinations thereof.Remodeling and/or reorganization can further involve compaction of thecells and/or tissue in the tissue construct. This second stage can becharacterized as further development of the tissue construct and canagain be guided by applying artificial parameters such as mechanicalconstraints, compression, shaking, electrical fields, magnetic fields,the action of objects embedded into the construct and may or may not besubjected to external forces, or gradients of morphogens and/or growthfactors. Typically, the combination of cellular aggregates of differentsizes in a stirred liquid promotes the formation of patternedarrangements. The combination of cellular aggregates of complementaryshapes promotes the formation of tissues with repetitive units.

Remodeling and/or reorganization can, for instance, involve applyinggeometrical constraints (such as using a chamber with specific geometry)to the tissue construct. This can induce self-organization into tissues(such as local compaction and local growth of capillaries).

The geometrical shape of the tissue in itself can induce localremodeling and/or reorganization of the cells, including compaction ofthe cells, local stress, and local sprouting of endothelial cells intoblood capillaries.

The cells can be assembled on chips made of biocompatible materialsincluding agarose, PDMS or PLLA cast on etched silicon wafers byconventional lithography process or replicated by hot-embossing.Polymers can be further functionalized to modify their interaction withcells by using coatings with polymers (e.g., PEG) or proteins (e.g.,BSA), or patterns of adhesive proteins promoting local adhesion of thetissue construct or nanometer and micrometer topographies. Chips are inthe order of centimeter scale and fit in classical cell-culturewell-plates. Wells in the order of 100 to 1500 μm (such as in the orderof 500 to 1500 μm) are generated in which the aggregates canself-assemble.

Depending on the methods that are used for the self-assembly of thecells and/or cellular aggregates, a wide variety of construct shapes canbe designed and prepared using the method of the disclosure. Forinstance, when using wells in which the aggregates are combined toconstructs, the shape of the wells will be translated to the shape ofthe construct.

In addition, the construct size may vary widely. However, the maximumsize may be limited by the diffusion distance of oxygen and nutrients. Away to overcome this is, for instance, by using perfusion or superfusionsystems. The constructs will normally have a size of at least 500 μm, orat least 1 mm. The upper limit of the size can, for instance, be 4 mm or1.5 cm.

When combining the cell aggregates to obtain a tissue construct,self-assembly may be assisted using a biomaterial, e.g., to form ascaffold and provide mechanical support or to assist in achieving aparticular desired shape. In addition, biomaterials or bio-activefactors may be included that guide the development or organization ofthe tissue construct. Types of biomaterials that can be incorporatedinclude, but are not limited to: ceramics, bioglasses, polymericmaterials (biodegradable or non-biodegradable), metals, and gels. Typesof bio-active factors that can be incorporated include, but are notlimited to: enzymes, receptors, neurotransmitters, hormones, cytokines,cell response modifiers such as growth factors and chemotactic factors,antibodies, vaccines, haptens, toxins, interferons, ribozymes,anti-sense agents, plasmids, DNA, and RNA. Biodegradable objects and/ormetallic objects are preferred. It is possible to combine the objectwith living cells, to combine the object with supracellular aggregates,and/or to combine the object with tissue constructs. The object can thusbe introduced in steps a) or b) and/or in steps c) or d). Metallicobjects can be used to modify the cellular aggregate or tissue by usingan electrical or magnetic field.

An important aspect of the disclosure is that the aggregates, afterhaving been combined, will self-assemble into biological tissues, whichmay vary in complexity. To this end, aggregates of different cell typesare preferably combined. Aggregates of the cell types that make up atissue may be combined to replicate the tissue. Features to incorporatein tissues may include, but are not limited to, a vascular network(endothelial cells and smooth muscle cells/pericytes), a neural network(neural cells), and a lymphatic network (lymphatic endothelial cells).For instance, for skeletal muscle tissue, aggregates of skeletal musclecells, neural cells, endothelial cells, smooth muscle cells/pericytes,and lymphatic endothelial cells may be combined.

Without wishing to be bound by theory, it is postulated that theself-assembly of the aggregates into tissue structures (also referred toas tissue morphogenesis) can be caused by migration of cells, physicaltraction or compaction of cells, local production of soluble orinsoluble (extra-cellular matrix) biological factors, differentiation ofcells, or combinations thereof.

The obtained tissue constructs can be used for various applications.They can, for instance, serve as a platform for creating constructs fortissue repair, or as a platform for studying tissue development (as ascientific tool), as an in vitro test model for compound testing inpharmacology or cosmetics, etc. The disclosure will now be furtherelucidated by way of the following, non-restrictive examples.

Examples

The micro-device shown in FIG. 2, consisting of an array of microwellsof various dimensions (well diameter, spacing and depth) fabricated inPDMS, was used for the spontaneous and simultaneous formation of anumber of microscale spheroids in a fast, controlled and reproducibleway (see FIG. 3). Aggregate formation is straightforward and requiresreduced amounts of cells and biological factors. The size of themicro-tissues is tunable (˜25 to 100,000 cells) and more suitable forimaging purposes. First, the optimal properties of the material werestudied, i.e., giving little or no cellular adherence and strongcellular aggregation for the preparation of spheroids based on hMSCs(human Mesenchymal Stem Cells) or HUVECs (Human Umbilical VeinEndothelial Cells), and the PDMS composition (curing agent:base ratio)in combination with various coatings was notably investigated. Bothparameters greatly influence cellular adherence and aggregation. Theresults range from strong to no adherence on the surface, and cellularassembly from isolate cell “suspension” to extensive cell aggregation.Best efficiency in the formation of spheroids is observed with a coatingof 35,000 MW PEG and a 10:0.5 PDMS composition. PDMS 10:0.5 gives thesmallest cellular adherence and 35,000 MW PEG at a concentration of 50mg/ml promotes cellular aggregation (see Table 1). The resultingspheroids exhibit a size in the hundreds of micron range depending onthe size of the microwells and the cell seeding density, see FIGS. 4Aand 4B.

Cellular aggregates can then be harvested and assembled into differentshapes, and different cell types can be combined. Here, the case of theassembly of hMSC and HUVEC aggregates into an agarose mold waspresented. The mold is made by replication of agarose on a stainlesssteel master (1×1×10 mm), see FIG. 5. Five thousand cellular aggregatesof each cell type were combined into this mold. They self-assembled intoa stratified tube with a layer of HUVEC surrounding a core of hMSC(FIGS. 7A and 7B). This self-assembly process is due to the differentialsurface tension of the two types of aggregates promoting segregation.Over time, the construct will remodel according to biological processesof angiogenesis and lead to a vascularized cylinder of dense tissue.

TABLE 1 Preparation of micro-tissues in coated PDMS-based microwells:Cellular aggregation and adherence on the surface depending on the PDMScomposition and the coating nature. Coating PDMS BSA BSA PEG 300 PEG35,000 composition Ø Fibronectin 10 mg/ml 50 mg/ml 10 mg/ml 50 mg/mlAgarose 10:0.5 Adherence: Adherence: Adherence: Adherence: Adherence:Adherence: Adherence: + + −−− +++ − No No Aggregation: Aggregation:Aggregation: Aggregation: Aggregation: Aggregation: Aggregation: + ++−−− ++ + +++ −−− 10:1 Adherence: Adherence: Adherence: Adherence:Adherence: Adherence: Adherence: −− − −−− +++ −−− + −−− Aggregation:Aggregation: Aggregation: Aggregation: Aggregation: Aggregation:Aggregation: −− +++ −− +++ − +++ −−− 10:3 Adherence: Adherence:Adherence: Adherence: Adherence: Adherence: Adherence: +++ +++ − +++ + +++ Aggregation: Aggregation: Aggregation: Aggregation: Aggregation:Aggregation: Aggregation: ++ + +++ + + + ++

In FIGS. 7A and 7B, human mesenchymal stem cell from bone marrow andhuman umbilical vein endothelial cells were separately cultured andaggregated onto chips. The chips are made of PDMS coated with 50 mg/mlBSA. The microwells on the chip are 200 microns diameter and 300 micronsdeep. Cells were allowed to aggregate into spheroids over 24 hours. hMSCare cultured in DMEM+glutamax, 100 nM dexamethasone (Sigma), 1%Pen/Strep (100 U/100 μg/ml, GIBCO), 50 mg/ml ITS-plus Premix (BD), 50μg/ml ascorbic acid (Sigma), 40 μg/ml proline (Sigma), 100 μg/ml sodiumpyruvate (Sigma). HUVEC are grown and aggregated in EGM2 medium (Lonza).

Five thousand spheroids of each cell type (10,000 spheroids total) weretransferred to an agarose chip with one trench (1 mm width, 1 mm depthand 1 cm long). This agarose (4%) is molded on a stainless steel mold.

The 10,000 spheroids quickly aggregated and formed a cylindrical tissueconstruct. This construct was cultured for six days and sectioned andimmunostained at days 3 and 6 for CD31 and Dapi.

A self-assembly of the two cell types in two concentric layers wasobserved at day 3 where the HUVEC are forming an external layer and thehMSC an internal core. This was followed by an invasion of the HUVECinto the center of the construct on day 6 and the formation of aprimitive capillary network.

In FIGS. 8A-8F, spheroids of hMSC were produced as described above.Fifteen thousand spheroids of 100 microns diameter were transferred ontoan agarose chip with wells of different shapes (i.e., squares, trianglesand circles). The agarose chip (4% agarose) is molded on a PDMS mold.The wells have a total surface area of 0.64 mm² and a depth of 1 mm. Thespheroids were seeded onto the chip and formed mesoscale tissue ofdefined size and shape. Those mesoscale tissues were harvested after 24hours and can be combined and used to build tissue models or tissueimplants.

FIG. 9A shows that the size of the building blocks depends on the numberof cells seeded and on the size of the microwells. Using humanmesenchymal stem cells and two different sizes of microwells (200 μm and400 μm), building blocks from 30 μm to 150 μm were assembled. In FIG.9B, it is also shown that different culturing media can induce differentlevels of compaction. Furthermore, as shown in FIG. 9C, different celltypes show different plasticity and maintenance of the shape over time.This plasticity decreased with longer incubation on the microwellsarray. For each cell type and each culturing medium, a time ofincubation on the microwells array has to be adjusted.

FIG. 10 shows that compaction of the tissue construct is not uniform forall shapes. Corners are regions of greater compaction. Compensatedshapes can be designed to promote remodeling of the tissue into adesired geometry.

In FIGS. 11A and 11B, it is shown that both the local compaction and thelocal stress of the tissue depend on the geometrical shape. FIG. 11Ashows a nuclear staining of 7 μm cuts. A local compaction of the cells(nuclei are closer to each other) was observed on the outside of tissuescompared to the inside in discs (left pictures) and tip effects withlocal cell compaction in the tips of triangular tissues (rightpictures). FIG. 11B shows a cytoskeleton staining of 7 μm cuts. Regionsof more intense F-actin populations of cells were observed. The shape ofthe tissues created local microenvironments of stress.

As can be seen from FIG. 12, further assembling into tissues ofclinically relevant size (such as centimeter-scale) is possible. Tissuesspontaneously fuse and can be manipulated, thus achieving clinicalrelevance.

What is claimed is:
 1. A method for in vitro producing a tissueconstruct comprising: a) combining living cells to form supracellularaggregates using spatial confinement; b) combining two or more of thesupracellular aggregates in a mold or on a biomaterial; c) applyingconditions that induce self-assembly within the combined supracellularaggregates to obtain the tissue construct; and d) applying conditionsthat induce tissue morphogenesis in the tissue construct.
 2. A methodaccording to claim 1, wherein said tissue morphogenesis comprisesmigration and/or differentiation of cells.
 3. A method according toclaim 1, wherein said spatial confinement comprises one selected from anarray of microwells, Hanging drop method, or microfluidic channels.
 4. Amethod according to claim 3, wherein said spatial confinement comprisesan array of microwells, said microwells having an enveloping diameter inthe range of 50-500 μm and a depth in the range of 100-1,000 μm.
 5. Amethod according to claim 3, wherein the microwells have a shape that isdifferent from a cylinder.
 6. A method according to claim 5, wherein theshape of at least some of the microwells is such that the resultingaggregates can self-assemble according to the lock-and-key principle. 7.A method according to claim 1, wherein 2-500,000 cells per microwell arecombined to form a supracellular aggregate, preferably 10-100,000 cellsper microwell, more preferably 10-10,000 cells per microwell.
 8. Amethod according to claim 1, wherein the living cells of the same ordifferent cell type are combined to form the supracellular aggregates.9. A method according to claim 1, wherein the cells are selected fromthe group consisting of endothelial cells, smooth muscle cells, striatedmuscle cells, neural cells, connective tissue cells, osteoblasts,osteoclasts, chondrocytes, hepatocytes, cardiomyocytes, myocytes,Schwann cells, urothelial cells, parenchymal cells, epithelial cells,exocrine secretory epithelial cells, epithelial absorptive cells,keratinizing epithelial cells, extracellular matrix secretion cells, orundifferentiated cells, such as embryonic cells, progenitor cells,(mesenchymal) stem cells, bone marrow cells, satellite cells,fibroblasts, and other precursor cells.
 10. A method according to claim1, wherein the supracellular aggregates have a mean particle size of20-400 μm as measured by light microscopy.
 11. A method according toclaim 1, wherein the biomaterial is selected from the group consistingof ceramics, (bio)glasses, polymeric materials (biodegradable ornon-biodegradable), and metals.
 12. A method according to claim 3,wherein the array of microwells is prepared by microchip technology, hotembossing, selective laser sintering, solid free-form fabrication, andphase separation micro-molding.
 13. A method according to claim 3,wherein the array of microwells comprises at least two microwells havinga substantially different size and/or shape.
 14. A method according toclaim 3, wherein the microwells are made of agarose, PEG(polyethyleneglycol) or PDMS.
 15. A method according to claim 3, whereinthe microwell surface is coated with one or more compounds capable ofreducing and/or preventing cellular adhesion, such as PEG, BSA, collagenand/or fibronectin.
 16. A method according to claim 1, wherein theliving cells are combined in the presence of fibronectin and/orcollagen.
 17. A method according to claim 1, wherein the surfaceproperties, the magnetic charge, and/or the electrical charge of thesupracellular aggregates are modified before combining two or more ofthe supracellular aggregates.
 18. A method according to claim 1, whereinthe supracellular aggregates are combined in a moving liquid, forinstance, a microfluidic chamber and channel.
 19. A method according toclaim 1, wherein the supracellular aggregates are combined in a wellhaving an enveloping diameter of at least 500 μm.
 20. A method accordingto claim 1, wherein the conditions in step c) comprise one or moreselected from mechanical constraints, compression, shaking, electricalfields, magnetic fields, and gradients of morphogens or growth factors.21. A method according to claim 1, wherein step d) comprises compactionof the cellular aggregates, preferably by applying geometricalconstraints to the tissue construct.
 22. A method according to claim 1,wherein in step a) or b) the living cells or the supracellular aggregateis combined with an object and/or wherein in step c) or d) the tissueconstruct is combined with an object, preferably a biodegradable objectand/or a metallic object, to induce a local response.